Fine-tuning of stable organic free-radical photosensitizers for photodynamic immunotherapy

Photodynamic immunotherapy (PDI) is an innovative approach to cancer treatment that utilizes photodynamic therapy (PDT) and photosensitizers (PSs) to induce immunogenic cell death (ICD). However, currently most commonly used PSs have restricted capabilities to generate reactive oxygen species (ROS) via a type-II mechanism under hypoxic environments, which limits their effectiveness in PDI. To overcome this, we propose a novel approach for constructing oxygen independent PSs based on stable organic free-radical molecules. By fine-tuning the characteristics of tris(2,4,6-trichlorophenyl)-methyl (TTM) radicals through the incorporation of electron-donating moieties, we successfully found that TTMIndoOMe could produce substantial amounts of ROS even in hypoxic environments. In vitro experiments showed that TTMIndoOMe could effectively produce O2˙−, kill tumor cells and trigger ICD. Moreover, in vivo experiments also demonstrated that TTMIndoOMe could further trigger anti-tumor immune response and exhibit a superior therapeutic effect compared with PDT alone. Our study offers a promising approach towards the development of next-generation PSs functioning efficiently even under hypoxic conditions and also paves the way for the creation of more effective PSs for PDI.


General Methods
All the chemicals were purchased from Innochem.Commercially available reagents were used without further purification.TTM was purchased from Pharmaron Beijing Co., Ltd.(China).Fluorescence emission spectra and full wavelength absorption spectra were performed on a 2300 EnSpire multimode plate reader.EPR spectra were performed on a Bruker ElexSys E580 spectrometer.The confocal laser scanning microscopic imaging studies were conducted with Leica TCS SP2 Confocal Microscope and Zeiss LSM880 Confocal Microscope.All 1 H NMR spectra were recorded at 400 MHz. 13 C NMR spectra were recorded at 100 MHz.HRMS were measured with a Thermo LCQ Deca XP Max mass spectrometer for ESI.MALDI-TOF mass spectra were recorded on a Bruker Autoflex III-MALDI-TOF-MS with DCTB as a matrix.Light sources (white light, 20 mW/cm 2 ) for chemical reactions and bioassays were from LED Light provided by PURI Materials, Shenzhen.The hypoxic environments in vitro experiments were constructed by SANYO O2/CO2 incubator MCO-5M, SANYO Electric Co., Ltd..The HPLC analysis was performed on the Thermo U3000, with chromatographic separation achieved on an InfinityLab Poroshell 120 EC-C18 column (150 mm × 4.6 mm, 4 μm particle size).A linear gradient from 50% B (0.1% formic acid in methanol) to 100% B (0.1% formic acid in methanol) in 5 min at a flow rate of 0.6 mL/min, and 100% B (0.1% formic acid in methanol) in 18 min at a flow rate of 0.6 mL/min with a detection wavelength of 600 nm was applied.Flow cytometric analysis of cells was conducted with BD FACSVERSE (BD Inc., USA).The human cancer cell lines HepG2 and A549 and mouse cancer cell line B16 in this study were from the Cell Resource Centre, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College.Dulbecco's modified eagle medium (DMEM), Roswell Park Memorial Institute (RPMI) 1640, and fetal bovine serum (FBS) were purchased from Gibco Company (USA).Penicillin and streptomycin were purchased from HyClone Company (USA).Anti-CD11c-APC, anti-CD86-PE, anti-CD4-FITC, anti-CD8a-PE, anti-CD25-APC, anti-FoxP3-PE, anti-MHC II-Percpcy5.5,anti-CD44-PE, and anti-CD62L-APC antibodies were all purchased from BioLegend, Inc. (San Diego, USA).Antibody for Calreticulin (CRT) was obtained from Abcam (Cambridge, UK).The ELISA kit for HMGB1 was purchased from Solarbio Science & Technology Co. Ltd (Beijing, China).The ELISA kit for ATP was obtained from Beyotime Biotech, Inc (Beijing, China).The antibody IFN-γ was purchased from Bioss Antibodies (Beijing, China).The ELISA Kits for IL-12p70, IL6, IFN-γ, and TNF-α detecting were purchased from Thermo Fisher Scientific (Waltham, MA, USA).The evaluation of biosafety was carried out by Mindray BC-5150.

Synthetic Procedures and Characterized Data
Scheme S1.Synthesis of TTMIndoOMe.

TTMIndo-1
In a 50 mL round bottom flask, TTM (553.3 mg, 1 mmol) was dissolved in 10 mL dry DMF.Then indole (351.5 mg, 3 mmol) and anhydrous Cs2CO3 (977.5 mg, 3 mmol) were added and the solution was stirred degassed (Ar) at 160 °C for 4 h in the dark.After cooling to r.t., the resulting mixture was poured into (1 M) hydrochloric acid solution, and the precipitate was filtered .The residue was purified by silica gel column chromatography (PE : DCM = 4 : 1) to give green solid 292.1 mg, yield 46%. 1

S8
In a 50 mL round bottom flask, TTMIndoNO2-1 (204.0 mg, 0.3 mmol) was dissolved in 5 mL THF, 5 mL EtOH and 2.5 mL H2O.Then NH4Cl (32.1 mg, 0.6 mmol) and Fe (33.6 mg, 0.6 mmol) were added and the solution was stirred at 80 o C overnight.After cooling to r.t., the resulting mixture was filtered with celite and filtrate was evaporated under vacuum.The residue was purified by silica gel column chromatography (PE : DCM = 1 : 4) to give green solid 171.6 mg, yielding 86%. 1

TTMIndoNHMe-1
In a 38 mL seal tube, TTMIndoNH2-1 (195.0 mg, 0.3 mmol) was dissolved in 5 mL dry DMF.Then K2CO3 (124.4 mg, 0.9 mmol) and CH3I (213.0 mg, 1.5 mmol) were added and the solution was stirred at 80 o C for overnight in the dark.After cooling to r.t., the resulting mixture was extracted by EA three times.Then, the organic phase was combined and dried by Na2SO4.The solvent was concentrated under vacuum and then residue was purified by silica gel column chromatography (PE : DCM = 2 : 1) to give green solid 155.4 mg, yield 78%. 1

UV-vis Absorption Spectra
The TTMIndo derivatives compounds were each dissolved in chloroform to generate stock solutions of 10 mM, respectively.
Immediately before UV-Vis measurement, aliquots of fluorogen stocks were diluted in chloroform, to obtain a final concentration of 10 μM.The TTMIndoOMe was each dissolved in DMSO to generate stock solutions of 10 mM, respectively.Immediately before UV-Vis measurement, aliquots of fluorogen stocks were diluted in PBS and water respectively, to obtain a final concentration of 20 μM.

DFT Calculations: Molecular Orbital Energy Levels
The DFT calculations were performed with the Gaussian16 series of programs [1] using the B3LYP hybrid functional and 6-31G(d) basis set.The spin densities and Mülliken spin densities were calculated by Multiwfn [2] .Molecular orbital energy levels were made with Multiwfn and VMD 1.9.3 software support.The isovalue of the figures are 0.03 au.

The Stability Evaluation of TTMIndoOMe in Different Cell Lysates
In the cell lysate, TTMIndoOMe was diluted to 100 μM in the lysate of B16, A549, and HepG2 cells and incubated at 37 °C for the specified time intervals.After incubation, twice the volume of methanol was added to the lysate, and the supernatant was obtained through centrifugation at 12000 rpm for 30 min.The HPLC system was performed on a Thermo U3000, with chromatographic separation achieved on an InfinityLab Poroshell 120 EC-C18 column (150 mm × 4.6 mm, 4 μm particle size).A linear gradient from 50% B (0.1% formic acid in methanol) to 100% B (0.1% formic acid in methanol) in 5 min at a flow rate of 0.6 mL/min, and 100% B (0.1% formic acid in methanol) in 18 min at a flow rate of 0.6 mL/min with a detection wavelength of 600 nm was applied.The HPLC chromatogram profiles of TTMIndoOMe, recorded at various incubation times, showed no degraded peaks or significant changes in peak areas over time, indicating the excellent stability of TTMIndoOMe within B16, A549, and HepG2 tumor cells as time extended.respectively.TTMIndoOMe was given at an equal dose of 100 μL, 15 mg/kg by subcutaneous injections at 6, 8, 10, 12, and 14 day.
After that, the tumors were treated with white light irradiation (20 mW/cm 2 ) for 15 min.The mice were anesthetized using isoflurane in the chamber, RWD small animal anesthesia machine.The white LED light were located about 2 cm below the chamber.Both mouse body weights and tumor volumes were recorded every two days during the whole treatment.The length (L) and width (W) of each tumor were measured with a caliper, and the tumor volume was calculated with the formula of V = (L × W 2 ) / 2. After 8 days of treatment, the mice were sacrificed and tumor tissue, spleen, and lymph nodes were harvested.The tumor tissue was taken for hematoxylin and eosin (H&E) and TUNEL (TdT-mediateddUTPNick-EndLabeling) stain.The spleen and lymph node were collected for flow cytometric analysis.

Antitumor Immune Response in vivo
Tumor-draining lymph nodes in each treatment group were obtained, then gently ground with rubbers of syringes and filtered to acquire single-cell suspension.Next, samples were centrifuged, and washed with PBS for 3 times followed by staining with Then, staining with anti-CD11c-APC, anti-MHCII-percpcy5.5, and anti-CD86-PE antibodies, the maturation of DC cells was examined by flow cytometry measurement.
Spleens in each treatment group were obtained, then gently ground with rubbers of syringes and filtered to acquire single-cell suspensions.Lysed with erythrocyte, and washed, single-cell suspensions of spleens in each treatment group were obtained.The percentages of CD3 + CD8 + T cells, and CD44 + CD62L + T memory cells were analyzed using flow cytometry.For intracellular staining, cells were fixed, permeabilized, and washed according to the manufacturer's protocol.The percentages of CD4 + CD25 + Foxp3 + Treg cells were analyzed using flow cytometry.
Tumor tissues were also used for IF and IHC analysis for characterizing CD8 + T cells and IFN-γ, H&E staining, and TUNEL staining.

Statistical Analysis
Statistical analysis was implemented using Student's t-test or one-way ANOVA.All the results were analyzed with the use of Prism 7 (Prism GraphPad Software, Inc., San Diego).Analysis of flow cytometry data was performed by FlowJo 7.6.1 software (BD Biosciences, New York, USA).Data were expressed as mean ± SD, mean ± SEM.

Figure S12 .S15Figure S13 .
Figure S12.ROS generation of TTMIndoOMe (a), and commercial PS methylene blue (b) and 2% DMSO as co-solvent (c) in PBS (λex = 480 nm) upon irradiation by the 20 mW/cm 2 white light LED irradiation ambient condition.O2 •-generation of of TTMIndoOMe (d), and commercial PS methylene blue (e) and 2% DMSO as co-solvent (f) in PBS (λex = 535 nm) upon irradiation by the 20 mW/cm 2 white light LED irradiation under ambient condition.

Figure S15 .
Figure S15.Stability study of TTMIndoOMe in the lysate of B16 cells at various time points (0 h, 0.5 h, 1 h, 2 h, 4 h, and 6 h) using HPLC analysis.

Figure S16 .
Figure S16.Stability study of TTMIndoOMe in the lysate of A549 cells at various time points (0 h, 0.5 h, 1 h, 2 h, 4 h, and 6 h) using HPLC analysis.

Figure S28 .Figure S29 .
Figure S28.Flow cytometric analysis of immune responses in tumor-bearing mice after indicated treatments.(a) Flow cytometric analysis of CD11c + CD86 + DCs cells, (b) CD11c + MHCII + DCs cells, and (c) CD86 + MHCII + DCs cells in the LNs of tumor-bearing mice after indicated treatments.(d) Flow cytometric analysis of CD3 + CD8 + T cells in tumor-bearing mice after indicated treatments.(e) Flow cytometric analysis of the Tregs (CD4 + CD25 + Foxp3 + T cells) in tumor-bearing mice.(f) Flow cytometric analysis of CD44 + CD62L + T cells in the spleen of tumor-bearing mice after indicated treatments.Data are given as the mean ± SEM (n = 6).in vivo.